We have selected the 10-step p-cymene pathway in Ps. putida PL as a prototype for pathways converging at "2,3-dihydroxybenzoates." We propose to (a) examine the specificity limitations to structural analogs as substrates, inducers and inhibitors of the pathway enzymes; (b) determine the regulatory units of the CYM pathway; (c) establish the ease, and mechanisms used for evolution to new catabolic potential and nutritional phenotype (acquisitive mutants); (d) subject the CYM genome to genetic analysis for evidence of gene clustering and gene order; (e) purify and characterize the four sequential enzymes (decarboxylase, hydrolase, hydratase, aldolase) that transform the ring cleavage product to pyruvate, and obtain their N-terminal sequences to see if they have evolved from a common ancestor; (f) examine the molecular nature and biological properties of the plasmids harbored by CYM strains, and clone the CYM pathway genes for in vitro mapping with restriction endonucleases. We propose to continue investigation of the enzymic mechanism used by bacteria for the catabolism of two classes of aromatic compounds. These are (i) resorcinylic compounds and (1,3-dihydroxybenzenes) and (ii) those compounds which are catabolized through "2,3-dihydroxybenzoates," as substrates for ring cleavage -i.e., 4-substituted toluenes. Some studies on the mechanism of flavoprotein hydroxylases, enzymes shown to indicate the catabolism of orcinol, resorcinol and 3-hydroxybenzoate in pseudomonads, will be completed; these include the stereospecificity of hydride transfer, the NIH shift, and a comparison of the properties of orcinol ad resorcinol hydroxylases isolated from the same strain of Ps. putida ORC. The ability of Azotobacter to grow with resorcinol will be studied, to confirm that pyrogallol is the intermediate used for ring cleavage: pyrogallol dioxygenase (ortho) and the oxalcrotonate branch (meta) enzymes will be studied with a view to understanding the possible origins of ortho- and meta-cleavage enzymes.